Dataset Information

An ?2,3-Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution.

ABSTRACT: Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the ?2,3-sialyltransferase from Photobacterium phosphoreum JT-ISH-467 (2,3SiaTpph ) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure-guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaTpph (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (?2,3-Neu5Ac-Lac) from lactose in a one-pot, two-enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new-to-nature sialoconjugates for biological studies.

SUBMITTER: Mertsch A

PROVIDER: S-EPMC7540698 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An α2,3-Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution.

Mertsch Alexander A He Ning N Yi Dong D Kickstein Michael M Fessner Wolf-Dieter WD

Chemistry (Weinheim an der Bergstrasse, Germany) 20200807 50

Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3-sialyltransferase from Photobacterium phosphoreum JT-ISH-467 (2,3SiaT<sub>pph</sub> ) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure-guided ...[more]

PMID: 32596832

Similar Datasets

Project description:Bacterial-bioluminescence regulation is often associated with quorum sensing. Indeed, many studies have been made on this subject and indicate that the expression of the light-emission-involved genes is density dependent. However, most of these studies have concerned two model species, Aliivibrio fischeri and Vibrio campbellii. Very few works have been done on bioluminescence regulation for the other bacterial genera. Yet, according to the large variety of habitats of luminous marine bacteria, it would not be surprising to find different light-regulation systems. In this study, we used Photobacterium phosphoreum ANT-2200, a piezophilic bioluminescent strain isolated from Mediterranean deep-sea waters (2200-m depth). To answer the question of whether or not the bioluminescence of P. phosphoreum ANT-2200 is under quorum-sensing control, we focused on the correlation between growth and light emission through physiological, genomic and, transcriptomic approaches. Unlike A. fischeri and V. campbellii, the light of P. phosphoreum ANT-2200 immediately increases from its initial level. Interestingly, the emitted light increases at much higher rate at the low cell density than it does for higher cell-density values. The expression level of the light-emission-involved genes stays constant all along the exponential growth phase. We also showed that, even when more light is produced, when the strain is cultivated at high hydrostatic pressure, no change in the transcription level of these genes can be detected. Through different experiments and approaches, our results clearly indicate that, under the tested conditions, the genes, directly involved in the bioluminescence in P. phosphoreum ANT-2200, are not controlled at a transcriptomic level. Quite obviously, these results demonstrate that the light emission of the strain is not density dependent, which means not under quorum-sensing control. Through this study, we point out that bacterial-bioluminescence regulation should not, from now on, be always linked with the quorum-sensing control.

Dataset Information

An ?2,3-Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution.

Publications

An α2,3-Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets